Study of the effects of the ovary temperature during transport on the oocyte a6er matura7on June 2016 Marta Rovira Rigau Introduc*on and Objec*ves During ovaries transport from the aba=oir to the laboratory, the oocytes in follicles undergo ischemic condi*ons. To alleviate adverse effects, *me of ovary storage must be as short as possible. However, when it is not possible, decreasing the ovary storage temperature would be an strategy to avoid ischemic damage. Un*l today, ovaries processed at the Bovine IVF Laboratory at the Veterinary College of UAB have been transported to the laboratory at 35-­‐37ºC and processed upon arrival. A new protocol is proposed which consists in storing the ovaries at room temperature and delaying the processing of the ovaries for 5 hours. The purposes of this study are: ü  To evaluate the effect of different storage protocols of bovine ovaries on oocyte quality measured by the assessment of the matura*on rate and the meio*c spindle configura*on aSer IVM. ü  To evaluate the effect of different storage protocols of bovine ovaries on the oocyte response to a vitrifica*on/warming process, by the assessment of the matura*on rate and the meio*c spindle configura*on. ü  To evaluate the effect of different storage protocols of equine ovaries on oocyte quality measured by the assessment of the nuclear matura*on rate. ü  To set up an op*mum transport and storage protocol for slaughterhouse ovaries that maximizes the quality of the collected oocytes, both in bovine and equine species. Materials and Methods Isothermal box 19-­‐22ºC Delayed aspira*on 5h IVM 24 h c e 1.  Formaldehyde 4% 2.  An*-­‐α-­‐tubulin monoclonal an*body 3.  An*-­‐Mouse IgG an*body – An*body-­‐
Alexa Fluor® 594 4.  DAPI Vitrifica*on and warming In vitro matura*on stages and meio*c spindle morphologies a Oocyte immunofluorescence Figure 1. Confocal laser-­‐scanning photomicrograph of bovine oocytes at different stages of in vitro b matura7on (a-­‐c) and altera7ons observed in the s p i n d l e m o r p h o l o g y a n d c h r o m o s o m e configura7on of bovine MII oocytes (d-­‐f). Oocytes were immunocytochemically stained to visualize the microtubules (blue) and counterstained to visualize chromosomes (red). a: Metaphase I d stage oocyte with microtubules having formed a clear meio*c spindle with chromosomes aligned at its equator. b: Anaphase I stage with chromosomes moving away from the the meio*c spindle equator. c: Normal barrel-­‐shaped metaphase II spindle with compact chromosomes arranged at the equator of the structure. d: Abnormal meio*c spindle f s t r u c t u r e s a s s o c i a t e d w i t h d i s o r g a n i z e d chromosomes. e: Cross sec*on of meio*c spindle with chromosomes par*ally dispersed and disorganized microtubules. d. Condensed and aligned chromosomes and absence of microtubules. Isothermal box Isothermal box 36-­‐38ºC 25-­‐30ºC 5h 35-­‐37ºC Ovary collec*on Delayed aspira*on Immediate aspira*on Immediate aspira*on Ovary collec*on Thermos IVM 30h 24 h Figure 2. Equine matured oocytes with the first polar body extruded (from Curcio et al., 2014) Results Effect of the ovary temperature during the transport on the matura7on rate and meio7c spindle of fresh bovine oocytes Chromosome morphology Microtubule morphology Treatment n MII, n (%) Normal Dispersed Decondensed Normal Dispersed Absent or absent 35-­‐37ºC 40 40 (100)a 30 (7,5)a 8 (20)a 2 (5)a 30 (75)a 8 (20)a 2 (5)a 19-­‐22ºC 188 87 (73,7)b 30 (34,5)b 56 (64,4)b 1 (1,1)b 30 (34,5)b 56 (64,4)b 1 (1,1)b Effect of the ovary temperature during the transport on the recovery rate and the nuclear matura7on rate of equine oocytes Bovine oocytes from Treatment n Total Recovery % Nuclear ovaries stored at oocyte rate matura7on room temperature 36-­‐38ºC 48 107 2,23 78,5a showed a lower 25-­‐30ºC 96 200 2,08 43,5b matura7on rate, Effect of the ovary temperature during the transport on the matura7on No s
ignificant d
ifferences w
ere o
bserved more a
bnormali7es rate and meio7c spindle of vitrified/warmed bovine oocytes of the meio7c spindle between treatments in the recovery rate Chromosome morphology Microtubule morphology of mare ovaries. and a worse response Treatment n MII, n (%) Normal Dispersed Decondensed Normal Dispersed Absent to vitrifica7on and Significant differences were observed or absent warming p
rocess between treatments in the nuclear 35-­‐37ºC 74 53(71,6)a 15(28,3)a 21 (39,6)a 17 (32,1)a 16 (30,2)a 17 (32,1)a 20 (37,7)a matura7on rate of equine oocytes. 19-­‐22ºC 48 26(54,2)b 2(7,7)b 19 (73,1)b 5 (19,2)b 2 (7,7)b 23 (88,5)b 1 (3,8)b Conclusions ü 
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Storing bovine ovaries at room temperature for 5 hours decreases the matura*on rate and increases abnormali*es in the meio*c spindle of oocytes. Response to vitrifica*on and warming is worse when the bovine ovaries are transported and stored for 5 hours at room temperature. Storing equine ovaries at room temperature for 5 hours does not affect the recovery rate but decreases the nuclear matura*on rate. The op*mum protocol for bovine and equine ovaries consists in processing them upon arrival at the laboratory. Reference: Curcio BR et al. Ultrastructural morphology and nuclear matura*on rates of immature equine oocytes vitrified with different solu*ons and exposure *mes. Journal of Equine Veterinary Science. 2014: 34(5), 632–640.